Plant transformation and exogenous protein expression is essential for molecular biology and biotechnology. Current approaches of stable plant transformation might be problematic and very time-consuming. Because of this, transient expression in protoplasts has become valuable alternative, being less cost and time-effective at the same time. Excellent for eukaryotic proteins, representing a natural cell habitat, protoplast isolation is widely used in protein interaction visualization techniques, like BiFC (Bimolecular fluorescence complementation) and FRET (Förster resonance energy transfer). In this protocol we present a another use of Arabidopsis protoplast in protein degradation assay, proving its high versatility as a tool in proteomics.

Adaxial surface of leaves was put on a piece of tape and leaves were flattened (Figure 1A-B).

Second piece of tape was applied with limited pressure, trapping
leaves between them (Figure 1C). To much pressure will result in
damaging leaves, too little pressure will lead to poor epidermis removal
effectiveness.

Sandwich was placed with adaxial surface on top
and pieces of tape were split by pulling away tape on top side, started
at the tip of leaf (Figure 1D-E) (see Note 2).

Tape was cut around leaves and put at room temperature
enzyme solution in Petri dish, exposed mesophyll down. Tape should not
be immersed in solution, but float on top of it. Mesophyll cells were
digested for 60 min at 30 °C with gentle shaking (55 rpm) (see Note 3).

After digestion pieces of tape were removed and cells were left for another 5 min in same conditions (see Note 4).

Using 10 ml pipette protoplasts were transported to 50 ml tubes and put on ice.

Protoplast were centrifuged for 3 min (150 x g, 4 °C) and washed
twice with W5 buffer. Be careful not to resuspend protoplast to
abruptly. After second wash step protoplast were resuspended in 1 ml of
MMg solution. Protoplasts were calculated with help of hemocythometer
and diluted to optimal concentration of 2 x 104 cells in 100 μl with MMg
solution (see Notes 5-7).

100 μl of isolated protoplasts in MMg medium were transferred to
the tube using a pipette tip with the tip of the tip cut off.

Using a pipette tip with the tip of the tip cut off 110 μl of PEG
solution was added. Solution was gently mixed and left for 15 min at RT
in horizontal position (see Note 9).

After incubation, 450 μl of W1
buffer was added to the tubes to dilute PEG solution, mixed and
centrifuged for 3 min, 300 x g.

Supernatant was removed and harvested protoplasts were resuspended in 300 μl of W1 solution.

Transfected protoplasts were incubated overnight at 22 °C in
horizontal position in the dark for protein expression. It is important
not to disturb protoplasts at this stage.

From this point
protoplasts can be used in various protein analysis techniques (kinase
assay, in vivo protein degradation assay, protein subcellular
localization, BiFC and FRET analyses) (Ludwikow et al., 2014). Here we
show in vivo degradation assay for plant proteins.

In vivo degradation assay in protoplasts

After overnight incubation (as indicated in step A14) in the dark
protoplasts were treated with 50 μM MG132 (proteasome inhibitor) or mock
treated with 0.1% DMSO for 6 h.

After brief centrifugation (300 x g, RT) supernatant was removed and cells were resuspended and
disrupted in 100 μl of protein isolation buffer.

Protein concentration was determined using a NanoDrop spectrophotometer.

Prepared samples were separated by SDS-PAGE and further analyzed by Western blotting.

Protoplasts isolated from WT Col-0 and the ABI1 knockout line (abi1td) were transformed with 5 µg of DNA plasmid coding for StrepTag-ACS6. Transformed protoplasts were treated with 50 µM MG132 or an equivalent volume of DMSO (mock control) for 6 h prior harvesting. A Western blot with anti-StrepTag antibodies confirms the presence of the StrepTag-ACS6 protein. The Western blot shown is representative of at least three independent experiments. Coomassie staining confirms equal protein loading (Ludwikow et al., 2014).

Notes

This protocol is applicable to Brassica napus protoplast isolation and transformation.

To avoid mesophyll cells damage (visible as dark green spots) don't use too much pressure when applying the tape.

It is not recommended to digest leaves for more than 60 min. Protoplasts yield at this point will not increase, but they lose viability.

If one hour digestion did not freed all mesophyll cells from tape fragments, one can gentle dip tape a few times in enzyme solution, to increase protoplast yield.

Use swinging bucket rotor for centrifugation.

Keep low acceleration and deceleration values during centrifugation.

Resuspend the protoplasts by gently rocking the tube.

Use hemocytometer to achieve accurate and reproducible results.

A large volume of plasmid DNA decreases transformation efficiency, therefore keep the volume around 10 μl. Low transformation efficiency is usually a result of low quality plasmid DNA.

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兰州大学

Department of Biotechnology, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University in Poznań, Poland

We succesfully adapted this protocol for rape and barley. Maybe protocol for barley protoplast isolation may be more sutable for rice. Here you go: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5078816/

Quaid-e-millath government college for women

Department of Biotechnology, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University in Poznań, Poland

Hi,
I think I don't fully understand your question. Part 'A' of this protocol covers all steps of isolation and transformation of Arabidopsis protoplasts. So yes, this protocol will help you identify isolated protoplasts under a microscope. Hope this answers your question.